Prion diseases are inevitably fatal infectious neurodegenerative zoonotic disorders of animals, including humans, with no known cure. Prions are comprised largely, if not entirely, of PrPSc, a misfolded form of the normal non-infectious prion protein PrPC. Although the details of how prions enter the nervous system and cause disease are not known the current model consists of prion trafficking to lymphoreticular tissues (LRS), replication/accumulation in these tissues and subsequent transport into the central nervous system (CNS). Neuroinvasion is thought to occur via sympathetic nerves that innervate LRS tissue and parasympathetic nerves that innervate the enteric nervous system in the gut wall. Recent work from our group and others has shown that prions that come into contact with mucosal surfaces spread to blood within minutes and can be detected in blood for the duration of disease. The role of this prionemia in pathogenesis, including neuroinvasion, has not been examined. We provide evidence that structures at the interface of the circulatory and nervous system can support prion replication and may be important portals of prion neuroinvasion. Overall, very little is known about the site(s) of initial establishment of infection, clearance of inoculum and whether replication of agent is required for neuroinvasion. This significant problem has been hampered by the inability to differentiate inoculum PrPSc vs. newly formed PrPSc. The long-term goal of these studies is to identify the sites of initial prion replication and to identify new routes of neuroinvasion. The objective of this application is to determine the temporal and spatial spread of inoculum PrPSc in the host following natural routes of inoculation to identify the pathways of prion spread and neuroinvasion. We hypothesize that prions rapidly spread throughout the host independent of agent replication. To accomplish this objective we have developed powerful animal systems and ultrasensitive prion detection techniques that have resulted in important findings in the initial uptake and spread of prions. Our proposed innovative approach does not require enrichment or labeling of PrPSc in trans, and avoids artifacts of altered PrPSc amyloid state and limited detection sensitivity. We have shown that PrPSc or PrPSc bound to soil crosses epithelia, drains to the lymphatic system and enters blood within minutes of oral or extranasal inoculation. We also show that once prions enter the peripheral nervous system they are retrogradely transported along defined neuroanatomical pathways. Finally, we have data indicating that a lymphoreticular system (LRS) replication-deficient prion strain is transported along known prion pathways but fails to establish disease. Using this new knowledge and techniques we determine if prionemia results in rapid widespread dissemination of prions, in the absence of prion conversion, which is followed by a tissue-specific rate of decay. The results of these aims will determine the early events in prion pathogenesis and if prion conversion is required for neuroinvasion and transynaptic spread.